Chromatography material and a method using same

ABSTRACT

A chromatography material for separation of nucleic acid mixtures, having a carrier and ion exchanger functions applied to it is described, whereby the carrier comprises a fibrous material. In addition, a method that is performed with the chromatography material according to this invention is also described.

[0001] The present invention relates to a chromatography material forseparation of nucleic acid mixtures and a method of separating nucleicacid mixtures using this chromatography material.

[0002] The use of chromatography and the chromatography materials usedin this process have become indispensable in such fields asbiochemistry, medicine, pharmacy and genetic engineering. With the helpof chromatography materials, biomolecules such as nucleic acids andproteins are rapidly and systematically separated and isolated. Inmolecular biology, it is often necessary to isolate certain nucleicacids, which are present in this mixture in concentrations of less than0.1%, from a naturally occurring mixture of more than a hundreddifferent components. The requirements of a chromatographic method andthe chromatography material used in the method therefore include, first,quantitative isolation of the nucleic acids, and secondly, quantitativeseparation of impurities to thereby purify the nucleic acid as amolecular species until it is homogeneous for subsequent analysis.

[0003] In the known chromatography methods, inorganic granularchromatography materials having defined particle and pore sizes areused, their surface having been modified with a silanizing reagent toproduce a stationary phase. The reaction with a reagent forming an anionor cation exchanger then leads to the finished chromatography material.

[0004] For example, International Patent WO 91/05606 describes a carriermaterial for chromatography which is suitable for separation of variousspecies of nucleic acids. Suitable carriers include silica gel, aluminumoxide, titanium dioxide, porous glass or resins. The carrier materialshould have a particle size of 3 to 500 μm with a pore size ofapproximately 10 to 1000 nm and a specific surface area of 5 to 800m²/g. The surface of this granular support is silanized withalkoxysilanes. Thus, in addition, a chromatographic carrier material isdescribed, which is based on silica gel and having anion exchangergroups that are obtained by reacting the alkoxysilane with a secondaryhydroxylamine.

[0005] Thus, according to European Patent 0 104 210, silica gelmaterials have a particle size of 3 to 100 μm, a void size of 10 to 1000nm and a specific surface area of 5 to 800 m²/g are described. Thesematerials are then treated at the surface with a silanizing agent andused with ion exchanger functions in chromatography for separation ofnucleic acids.

[0006] Finally, European Patent 0744025 discloses a chromatographymaterial in which a carrier of silica gel is reacted with a silanizingreagent, whereby carrier materials having a pore diameter of 4 to 6 nmare selected. The particle size of the carrier is 1 to 500 μm.

[0007] In chromatographic separation of nucleic acid mixtures usingconventional granular chromatography materials, it has been found thatit is very time-consuming to perform such separations. For example, if acertain column was used with a modified silica gel as the carrier forchromatography, it would be expected that an adsorption time of thenucleic acids on the carrier of at least 20 minutes under gravity flowconditions would have to be accepted.

[0008] The chromatography materials known in the past have also had adefined porosity, because according to the prevailing opinion only aporous support, i.e., one with an enlarged surface area, would guaranteesufficient loading with ion exchanger. However, this porosity has thedisadvantage that the quality of nucleic acid separation is not optimum.This is attributed to the fact that other substances present in themixture during separation, e.g., RNA and proteins, diffuse into thepores and thus have a negative effect on the separation process.

[0009] Thus, the object of the present invention was to make available achromatography material that could perform a separation of nucleic acidmixtures within the shortest possible period of time, while at the sametime achieving an excellent resolution of the nucleic acid mixtures andthus an excellent purity of the nucleic acids isolated.

[0010] Another object of the present invention is to make available amethod with which a nucleic acid mixture can be separated with thehighest possible resolution and purity of the individual components inthe shortest possible amount of time.

[0011] These objects are achieved with the features of Patent claim 1and Patent claim 12.

[0012] The present invention relates to a chromatography material forseparating nucleic acid mixtures, having a carrier and ion exchangerfunctions applied to it, wherein the carrier is a fibrous material.

[0013] The subclaims relate to preferred embodiments of thechromatography material according to this invention.

[0014] This invention relates to a method of separating nucleic acidmixtures with the chromatography material according to this invention,where the chromatographic separation of the nucleic acids is performedby the action of a force.

[0015] The subclaims concern preferred embodiments of the methodaccording to this invention.

[0016] Finally, this invention concerns a kit for separating nucleicacid mixtures which contains a chromatography material according to thepresent invention. The kit includes the chromatography materialaccording to this invention in the desired arrangement together withcorresponding buffers for performing the chromatographic separation ofnucleic acid mixtures using the chromatographic material.

[0017] This invention is explained on the basis of the figures, whichshow:

[0018]FIG. 1 an example of a column using the chromatographic materialaccording to this invention;

[0019]FIG. 2 separation of nucleic acid mixture components on an agarosegel using the chromatography material according to this invention;

[0020]FIG. 3 separation of nucleic acid mixture components on an agarosegel using a state-of-the-art chromatography material, and

[0021]FIG. 4 separation of nucleic acid mixture components on an agarosegel using a state-of-the-art chromatography material.

[0022] It has surprisingly been found according to this invention that asignificantly improved separation capacity can be achieved if thecarrier of the chromatography material includes a fibrous material whosesurface area is not enlarged or is only insignificantly enlarged.Contrary to the prevailing opinion, the chromatography materialaccording to this invention may be loaded with a corresponding amount offunctional ion exchanger groups to guarantee an excellent separationcapacity.

[0023] The specific surface area of the carrier is in the range of 0.05to 50 m²/g, preferably in the range of 1 to 10 m²/g, in particular 1 to5 m²/g.

[0024] Fibrous materials suitable for modification of their surface areawith ion exchanger functions may be used as the carrier. A suitablematerial for the carrier would be, for example, micro-fibers, wherebymicro-glass fibers are preferred. Such materials have a pore-freesurface.

[0025] In a preferred embodiment, the fibrous material may be subjectedto an acid or base treatment before being used as a carrier.

[0026] The fibrous carrier may be in the form of a mass which may beused directly for chromatography. However, it is also possible toprocess the mass further for separation into suitable forms.

[0027] It has been found that for many applications the chromatographymaterial according to this invention advantageously includes a carrierwhich is in the form of a membrane. For a suitable capacity of thechromatography material, it is preferable for the membrane to have athickness of at least 0.05 mm, regardless of the total surface area.

[0028] In a preferred embodiment of the chromatography materialaccording to this invention, the membrane is in the form of a singlelayer. However, it is also possible for the membrane to be in multiplelayers, depending on the desired separation capacity.

[0029] The carrier of the chromatography material according to thisinvention is reacted with a silanizing reagent. For example, thesilanizing reagent may be the one described in International Patent WO91/05606. Likewise, anion exchanger groups or cation exchanger groupsmay be applied to the stationary phase by known methods. An example ofthis is described in International Patent WO 91/05606.

[0030] The chromatography material according to this invention has theadvantage that when using this material, it is possible to perform aseparation of nucleic acid mixtures within an extremely short period oftime. The reason for this is that there is a sufficient retention timefor separation of the nucleic acids even in the presence of strongforces, e.g., a vacuum acting on the chromatography material accordingto this invention, which has a higher material density than traditionalchromatography materials. Because of the high material density that canbe achieved, separation of nucleic acids may be performed with a verysmall bed volume. The washing and elution volumes are also lowaccordingly.

[0031] The chromatography material according to this invention is usedto separate nucleic acid mixtures with an extremely high accuracy andpurity of the fractions to be obtained. This is manifested in particularby comparison with the traditional granular chromatography materials. Tothis end, the nucleic acid mixtures were allowed to run on an agarosegel on the chromatography material according to this invention and twotraditional chromatography materials both before and after separationinto RNA and DNA.

[0032]FIG. 2 shows the separation of the nucleic acid mixture componentsusing the chromatography material according to this invention. Theindividual lanes show the runs through the column:

[0033] Lane L: cleared lysate before separation

[0034] Lane D: column run

[0035] Lane W 1: first washing

[0036] Lane W 2: second washing

[0037] Lane E: elution

[0038] The same experiment was conducted with traditional chromatographymaterials. To do so, reference is made to FIGS. 3 and 4. The same columnruns were applied in the tracks.

[0039]FIG. 2 shows that in the eluate in lane E, the RNA is completelyseparated from the plasmid DNA. In addition, it can be seen clearly thatthe individual runs before elution do not entail any loss of DNA.

[0040]FIGS. 3 and 4 show the separation of nucleic acid mixturecomponents using traditional granular chromatography materials. In thepurification in FIG. 3 in particular, a great loss of DNA is indicatedin lane E. Furthermore, there is a poor reduction in concentration ofRNA, which is shown in lane W 2, where significant quantities of RNAcontinue to be present in the second washing run.

[0041] A poor reduction in concentration is also obtained when usinganother traditional chromatography material in FIG. 4. Lane W 2 showsconsiderable RNA still present in the second washing.

[0042] Another advantage in comparison with traditional granularchromatography materials is that no particles of the chromatographymaterial (fines) are present in the eluate E. This leads to aconsiderable improvement in the quality of the nucleic acids thusobtained.

[0043] The method according to this invention for separating nucleicacid mixtures using the chromatography material according to thisinvention is characterized in that it is performed under the influenceof a force.

[0044] In a preferred embodiment, the method is performed by applying avacuum.

[0045] For example, after application of the nucleic acid mixture sampleto the chromatography material according to this invention, a vacuum isapplied, inducing separation within a approximately twenty seconds. Thisis in gross contrast with the traditional silica gel columns whichrequire a separation time of at least twenty minutes with gravity flowwhen using a bed volume at least ten times greater.

[0046] The chromatography element according to this invention may beused in virtually all chromatographic processes. These include columnchromatography, separation in spin columns and spin cups or separationin batch processes, where the chromatography material is in suspensionor is adsorbed on reaction vessels, microtiter plates, pipette tips,stirring rods or test strips.

[0047] In chromatographic separation in spin cups, the centrifugal forceis utilized in that the specimen which is placed in spin cups isseparated chromatographically in a centrifuge.

[0048] Any nucleic acid mixture can be separated very effectively byusing the chromatography material according to this invention. It isthus possible to isolate DNA with an extremely high purity from mixturescontaining only very small quantities of DNA in addition to largequantities of RNA. Furthermore, it is possible to completely avoid usingtoxic substances such as phenol, chloroform or ethidium bromide. Inaddition, the separation may be performed entirely without the use ofRNAse.

[0049]FIG. 1 shows an example of a column equipped with thechromatography material according to this invention. A bottom frit (2)having a thickness of 1 mm sealed with the outlet is provided in aconventional commercial plastic column (1) having an outlet that taperstoward the bottom for applying a vacuum. The chromatography material (3)according to this invention in the form of a micro-glass fiber membranewith a thickness of 2 mm is applied to this. In conclusion, a top frit(4) 1 mm thick is provided on top of that.

[0050] Similar arrangements are used in all other chromatographymethods, e.g., in spin cups and on microtiter plates (96-well plates).

[0051] The method according to this invention for separating nucleicacid mixtures using the chromatography material according to thisinvention is carried out in a simple step gradient by washing thearrangement loaded with the nucleic acid mixture and then eluting thedesired nucleic acid with a suitable buffered salt solution. Most of theRNA is separated during binding of the DNA to the chromatographymaterial according to this invention, and the remaining RNA is washedout during the washing operation. Thus, a treatment with RNAse is notnecessary.

[0052] If the chromatographic separation is carried in a column or amicrotiter plate, for example, then separation is performed on thearrangement of components by applying a vacuum within an extremely shortperiod of time, i.e., in approximately twenty seconds. Likewise,separation with an excellent efficiency is achieved in spin cups whichare placed in a centrifuge, and then the separation is performed bycentrifugal force within a very short period of time, such asapproximately twenty seconds.

[0053] The chromatography material according to this invention may bebrought on the market in various ways. For example, it is possible tooffer the chromatography material according to this invention in thedesired arrangement as a kit together with the equipment required forthe chromatography, e.g., buffers. Other commercial forms are of coursealso included.

[0054] This invention is illustrated now on the basis of the followingexamples, although without being restricted to them.

EXAMPLES Example 1 Culturing the Bacterial Cultures

[0055]E. coli cultures are cultured according to conventionalmicrobiological practice for the plasmid preparations. On day 1, anisolation smear is prepared from a deep-frozen stock culture on aselective medium (e.g., LB agar with ampicillin as the antibiotic).After incubation overnight at 37° C., a well grown single colony isinoculated on 50 to 300 ml liquid medium (e.g., LB) to which thecorresponding antibiotic has been added on day 2. After anotherovernight incubation at 37° C. on a shaker with good ventilation (200 to300 rpm), even larger volumes of culture are optionally stocked up orthe culture that has been grown is harvested directly. In the case ofstocking up, the corresponding amount of fresh liquid medium mixed withthe respective antibiotic is inoculated with the culture of thepreceding day in the amount of 1% of its volume and incubated on theshaker at 200 to 300 rpm for another overnight incubation at 37° C.

[0056] For a mini-preparation, 1 to 3 ml culture with high-copy plasmidor 5 to 20 ml culture with low-copy plasmid is used. In the case ofmidi- or maxi-preparations, larger amounts are used accordingly.

Example 2 Isolation of DNA from Bacteria

[0057] The amount of bacterial culture indicated in Example 1 for amini-preparation is centrifuged for three minutes at 13,000×g in asuitable centrifuge vessel, and the supernatant medium is discardedcompletely. Any medium running back from the edge of the centrifugevessel is removed with a pipette and also discarded.

[0058] The pelletized bacteria are completely re-suspended by vortexingin 0.4 ml buffer at 50 mM Tris-HCl (pH 8.0)/10 mM EDTA/100 μg/ml RNAse.There must not be any visible cell clumps or cell aggregates.

[0059] The suspended cells are lysed by adding 0.4 ml buffer of 200 mMNaOH/0.1% (w/v) SDS. The suspended cells are mixed with the lysis bufferby inverting several times until forming a homogeneous phase. This phasehas a very high viscosity due to the genomic bacterial DNA emerged. Itis incubated for a maximum of five minutes at room temperature.

[0060] The lysis mixture is neutralized by adding 0.4 ml buffer of3.1-3.4 M potassium acetate (pH 5.5 with acetic acid). After adding thebuffer, the mixture is blended by inverting repeatedly until obtaining ahomogeneous phase. This phase then has a low viscosity again. There mustnot be any viscous residues of cell lysate.

[0061] The precipitate of bacterial proteins and cell debrisprecipitated in neutralization is centrifuged by centrifuging for 10minutes at ≧13,000×g at room temperature, and the clear supernatant(“cleared lysate”) is pipetted out.

Example 3 Producing a Column Using the Chromatography Material Accordingto this Invention

[0062] 5 g pore-free glass fibers having a specific surface area of 5m²/g is mixed with 60 g 3-glycidoxypropyltri-methoxysilane and 0.13 mltriethylamine in 750 ml dry xylene. The reaction mixture is degassed byapplying a vacuum three times and then aerating with nitrogen and nextheating for four hours at 130° C. in the absence of air and moisture.The mixture is filtered and washed with xylene and tetrahydrofuran. Themodified glass fiber is dried in vacuo at 50° C.

[0063] The product is then mixed with 750 ml and 42 g diethylamine andheated for 18 hours at reflux. The product is washed with dioxane andmethanol and dried at 70° C. in vacuo. The modified glass fiber masshaving an anion exchanger function is then processed further to form ananion exchanger membrane. The glass fiber mass is slurried in acetoneand rolled into the proper form or cast and then dried. A membrane iscut to conform to the column diameter and inserted into the arrangementaccording to FIG. 1.

Example 4 Separation of the Bacterial DNA

[0064] The column according to example 3 is connected to a suitablevacuum chamber (e.g., VacMan, Promega). The ion exchanger membrane isequilibrated with 2 mL buffer of 100 mM NaAc/HAc (pH 5.0)/600 mM NaCl.To do so, the buffer is pipetted into the column and pulled completelythrough the membrane by applying a water jet vacuum. The vacuum pumpremains turned on until no liquid is being detectably sucked away fromthe membrane. Then the vacuum is switched off.

[0065] The column is loaded with the cleared lysate from Example 2,which is drawn completely through the membrane by applying the water jetvacuum. The vacuum pump remains in operation until it is apparent thatno more liquid is being removed from the membrane. Then the vacuum isturned off.

[0066] To remove non specifically bound components, the column is washedwith 2.5 ml buffer of 100 mM NaAc/HAc (pH 5.0)/600 mM NaCl. To do so,the buffer is pipetted into the column and drawn completely through themembrane by applying a water jet vacuum. The vacuum pump remains turnedon until it is apparent that no more liquid is being removed from thematrix. Then the vacuum is turned off. This washing step is repeatedonce in the case of the mini- and midi-preparation.

[0067] The column is detached from the vacuum chamber and the plasma DNAbound to the membrane is eluted directly into a suitable vessel byadding 0.8 ml buffer of 100 mM Tris-HCl (pH 8.5)/1250 mM NaCl. To do so,the buffer is pipetted into the column and forced manually through themembrane with the help of a suitable stamp. To do so, the elution buffershould be forced through in a rapid sequence of drops, but by no meansas a stream. Individual droplets must still be clearly discernible withthe naked eye.

[0068] The eluates are mixed with 0.7 vol isopropanol (room temperature)and mixed well. The plasma DNA precipitated in this way is centrifugedfor 30 minutes at ≧13,000×g and 4° C. and the supernatant is discarded.The pelletized DNA is washed once with 80% ethanol, centrifuged again,then dried (either by leaving to stand at room temperature or in vacuo)and the dried DNA is finally dissolved in a suitable amount of TE bufferor water for ten minutes at 37° C.

[0069] The dissolved plasmid DNA is measured by spectrophotometry andanalyzed on an agarose gel.

[0070]FIG. 2 shows the separation of the components of the mixture on a1% agarose gel using a TAE buffer pH 8.3. The following components wereapplied in the individual lanes:

[0071] Lane L: cleared lysate before separation

[0072] Lane D: column run

[0073] Lane W1: first washing

[0074] Lane W2: second washing

[0075] Lane E: elution

[0076] The lanes L, D and W1 show clearly that RNA is still present inthe preparation. However, complete separation of the nucleic acidmixture components is clearly apparent in the eluate in lane E whichshows only plasmid DNA without any RNA contamination.

Example 5 Separation of Bacterial DNA

[0077] The same bacterial DNA as in Example 2 was applied to a columnfrom Macherey-Nagel (Nucleobond Kits) containing a traditionalchromatography material and separated according to the manufacturer'sinstructions. The results are shown in FIG. 3.

Example 6 Separation of Bacterial DNA

[0078] A traditional chromatography material from the company Qiagen(Qiagen Plasmid Kits) was used to separate the bacteria of Example 2.The manufacturer's instructions were also followed here. The results areshown in FIG. 4.

1. A chromatography material for separating nucleic acid mixtures,having a carrier and ion exchanger functions applied to it,characterized in that the carrier is composed of micro-glass fibers. 2.The chromatography material according to claim 1, characterized in thatthe specific surface area of the carrier amounts to 0.05 to 50 m²/g. 3.The chromatography material according to at least one of claims 1 and/or2, characterized in that the fibrous material is in the form of a mass.4. The chromatography material according to claim 3, characterized inthat the mass is designed as a membrane.
 5. The chromatography materialaccording to claim 4, characterized in that the membrane has a thicknessof at least 0.05 mm.
 6. The chromatography material according to claim5, characterized in that the membrane is in the form of a single layer.7. The chromatography material according to claim 6, characterized inthat the membrane is in the form of multiple layers.
 8. Thechromatography material according to at least one of claims 1 through 7,characterized in that the carrier is reacted with a silanizing reagent.9. The chromatography material according to claim 8, characterized inthat anion exchanger groups are linked by the silanizing reagent. 10.The chromatography material according to claim 8, characterized in thatcation exchanger groups are linked by the silanizing reagent.
 11. Amethod of separating nucleic acid mixtures using a chromatographymaterial according to at least one of claims 1 through 10, characterizedin that the chromatographic separation is performed by the applicationof a force.
 12. The method according to claim 11, characterized in thata vacuum is applied.
 13. The method according to claims 11 and 12,characterized in that the chromatographic separation is performed in acolumn or on microtiter plates.
 14. The method according to claim 13,characterized in that the separation is performed by centrifugal force.15. The method according to claim 14, characterized in that theseparation is performed in spin cups in the centrifuge.
 16. The methodaccording to at least one of claims 11 through 15, characterized in thatthe chromatographic separation is performed in a step gradient.
 17. Themethod according to at least one of claims 11 through 16, characterizedin that no RNAse is used.
 18. A kit for separating nucleic acid mixturescontaining a chromatography material according to at least one of claims1 through 10.